It has been shown that a combination of heparin or a hexasaccharide fragment together with a suitable steroid cause inhibition of angiogenesis in mammals and that tumor masses in mammals are caused to regress and metastasis is prevented (Folkman et al.; Science 1983, 221, 719-725). It was also shown that this combination was effectively inhibiting other angiogenesis depending processes such as fertilization of the rat (Folkman et al., European Patent Application No. EP-0114589). It is also effective in reducing osteoporosis and in treating deseases involving neovascularization, such as neovascular diseases of the eye. Also because of the occurrence of angiogenesis in psoriasis and arthritis, it is expected that this combination will be useful in treating those diseases.
Whereas further research and development with respect to the steroid part of this combination that inhibits angiogenesis has yielded more potent and specific steroids (Crum et al., Science 1985, 230, 1375-1378), no such progress has been made with regards to the heparin component of the heparin steroid composition. A synthetic highly sulfated non-anticoagulant pentasaccharide was recently shown to possess inhibiting effect on angiogenesis in the presence of a steroid to the same extent as its highly anticoagulant active analogue (the 3-O-sulfated analogue) (Choay et al., European Patent Application No. EP-0140781). This finding further supports the finding that the anticoagoulant (anti-Xa) properties of heparin and heparin fragments are not required for the heparin and heparin fragments to be inhibitory on angiogenesis in the presence of steroids (Folkman et al., Science 1983, 221, 719-725; Crum et al., Science 1985, 230, 1375-1378).
Heparin is a glycosidically linked highly sulfated copolymer of uronic acids and D-glucosamine. The uronic acids being L-iduronic acid or D-glucuronic acid of which the former usually is sulfated and the latter usually nonsulfated. The glucosamine is either N-sulfated or N-acetylated and also frequently 6-O-sulfated. Small amounts of other structural variants also occur. The exact structure of heparin and the precise nature of its antithrombotic mechanism of action has not been elucidated although it has been in widespread use for almost 50 years. Heparin is polydisperse with a molecular weight range from 3,000-30,000 with many structural variations within a given chain. The exact composition of heparin varies depending on its source, which usually is porcine intestinal mucosa, bovine lung, bovine intestinal mucosa, or ovine intestinal mucosa and also depending on the method for its preparation and purification. Low molecular weight heparin (molecular weight range 2,000-10,000) has been isolated in small amounts by fractionation of standard heparin. Heparin fragments of molecular weight range 500-10,000 has been prepared by partial depolymerization of heparin by chemical or enzymatic methods. Chemical depolymerization has been carried out in many different ways, frequently by nitrites at low pH, by alkaline .beta.-elimination usually after esterification of uronic acids or by oxidative methods usually using peroxides or periodate. After depolymerization with nitrites the newly formed anhydromannose at the reducing end of the heparin fragments and the oligosaccharides derived from such a fragment usually are reduced to anhydromannitol or oxidized to anhydromannonic acid for increased stability of the product. The enzymatic depolymerization and the alkaline .beta.-elimination results in the same 4,5-unsaturation in the nonreducing end of the heparin fragments and in the oligosaccharides derived from these fragments. For increased stability such unsaturated groups can subsequently be reduced by standard procedures for example catalytic hydrogenation, or the whole 4,5-unsaturated monosaccharide may be eliminated by for example mild acid treatment or by applying metal containing reagents such as mercury salts. In the latter case heparin fragments, heparan sulfate fragments, and oligosaccharides derived from them containing an uneven number of saccharide moieties are obtained. Heparan sulfate is the only other glycosaminoglycan besides heparin that also contains N-sulfated glucosamines. Most heparan sulfate however contains more N-acetylated glucosamine than N-sulfated glucosamine, the opposite being the case for heparin.
The same methods of fractionation and depolymerization used for heparin are also applicable to heparan sulfate. The enzyme used for heparan sulfate is usually a hepartitinase (heparanase) instead of heparinase which is most commonly used for heparin. Small amounts of heparan sulfate is usually found in standard heparins. Heparan sulfate also constitutes a large part of heparin by-products particularly form bovine lung.
Heparin by itself (without a steroid) enhances the intensity of angiogenesis induced by tumors and by tumor derived factors in vivo, although in the absence of tumor cells or tumor extracts or tumor derived factors neither heparin nor the mast cells which release heparin could induce angiogenesis (Taylor and Folkman, Nature 1982, 297, 307-312).
Some angiogenic factors from normal cells and tissue, for example so-called heparin binding growth factors can also induce angiogenesis and stimulate the growth of capillary endothelial cells. With some growth factors, this stimulation of capillary endothelial cell growth is potentiated by heparin.
Heparin, by virtue of its high negative charge, has a strong affinity for cations, where the binding generally is ionic since pH dependency is usually observed. Clinically used standard heparin is either the sodium or calcium salt of heparinic acid. The calcium heparin usually has a calcium content of about 11 w/w % which corresponds to about 2.8 .mu.mole Ca.sup.2+ /mg heparin. For cupric ions (Cu.sup.2+), binding to heparin was shown to be pH-dependent and for a typical heparin (molecular weight 13100, anticoagulant activity 146 IU/mg) copper binding was 0.606 .mu.mole Cu.sup.2+ /mg heparin at neutral pH (Stivala SS, Fed. Proc. Fed. Am. Soc. Exp. Bio. 1977, 36, 83-88). Thus cupric ions bind to a lesser extent to heparin than calcium ions at neutral pH, showing for cupric ions about 20% of the binding of that of the calcium ions. Heparin containing 1 .mu.g of copper per 20 .mu.g of heparin (which corresponds to 0.787 .mu.mole Cu.sup.2+ /mg heparin or about 10 .mu.mole Cu.sup.2+ /.mu.mole heparin) has been shown to be able to induce angiogenesis in vivo, which heparin by itself could not do (Alessandri G. Raju K, Gullino P M, Microcirculation, Endothelium and Lymphatics 1984, 1, 329-346).
Metal chelate affinity chromatography, also called ligand exchange chromatography or immobilized metal ion adsorption, is usually carried out by binding (chelating) various metal ions, such as for example Cu.sup.2+, Zn.sup.2+, Ni.sup.2+, Co.sup.2+, Mn.sup.2+, Ca.sup.2+, Fe.sup.2+ and Fe.sup.3+ to a solid matrix, for example a cation exchange resin or a special metal ion chelator such as Chelating Sepharose.RTM. 6B Pharmacia and Chelex.TM. 100 Bio-Rad and then carry out fractionation of complex mixtures.